Caspase Domain Duplication During the Evolution of Caspase-16

Abstract

Caspases are cysteine-dependent aspartate-directed proteases which have critical functions in programmed cell death and inflammation. Their catalytic activity depends on a catalytic dyad of cysteine and histidine within a characteristic protein fold, the so-called caspase domain. Here, we investigated the evolution of caspase-16 (CASP16), an enigmatic member of the caspase family, for which only a partial human gene had been reported previously. The presence of CASP16 orthologs in placental mammals, marsupials and monotremes suggests that caspase-16 originated prior to the divergence of the main phylogenetic clades of mammals. Caspase-16 proteins of various species contain a carboxy-terminal caspase domain and an amino-terminal prodomain predicted to fold into a caspase domain-like structure, which is a unique feature among caspases known so far. Comparative sequence analysis indicates that the prodomain of caspase-16 has evolved by the duplication of exons encoding the caspase domain, whereby the catalytic site was lost in the amino-terminal domain and conserved in the carboxy-terminal domain of caspase-16. The murine and human orthologs of CASP16 contain frameshift mutations and therefore represent pseudogenes (CASP16P). CASP16 of the chimpanzee displays more than 98% nucleotide sequence identity with the human CASP16P gene but, like CASP16 genes of other primates, has an intact protein coding sequence. We conclude that caspase-16 structurally differs from other mammalian caspases, and the pseudogenization of CASP16 distinguishes humans from their phylogenetically closest relatives.

Keywords: Caspase; Evolution; Protein domain; Pseudogenization; Pyroptosis.

Fig. 1

Caspase-16 contains a catalytic site that is conserved in species of the main clades of mammals. Amino acid sequence alignment of caspase-16 proteins of the chimpanzee (Pan troglodytes) (GenBank accession number: XP_523278.4), cattle (Bos taurus) (XP_005224700.2), opossum (Monodelphis domestica) (XP_016279605.1) and echidna (Tachyglossus aculeatus). The caspase domain is indicated by a red line above the sequences. Residues conserved in all or more than half of the sequences are highlighted by red and blue fonts, respectively. Yellow shading marks histidine and cysteine of the catalytic dyad (Color figure online)

Fig. 2

Caspase-16 is phylogenetically closely related to caspase-14. The caspases (CASPs) of chimpanzee (Pan troglodytes, Pt), cattle (Bos taurus, Bt), opossum (Monodelphis domestica, Md) and echidna (Tachyglossus aculeatus, Ta) were subjected to maximum likelihood analysis based on the amino acid sequence alignment of the caspase domain. Bootstrap values above 80 are shown on the cladogram. Paralogs of CASP14 of the echidna are named CASP14-like (CASP14L) 1 through 4, and paralogs of CASP1 of the opossum are named CASP1-like (CASP1L) 1 through 3. On the right, the main clades of caspases are labelled according to the best characterized member of each clade (e.g. CASP1-likes)

Fig. 3

Exon duplications have led to the duplication of the caspase domain in caspase-16. A Schematic depiction of domains of the caspase-16 protein and exons of the corresponding gene. The model for evolution of exons is indicated by arrows from a putative ancestral CASP gene (assuming the same exon–intron structure as in CASP1) to CASP16. B Alignment of amino acid sequences encoded by different exons of chimpanzee CASP16. Red fonts highlight identical residues. Dashes were introduced to optimize the alignments. C, D Comparison of structure models of a human caspase-14 dimer and macaque caspase-16. Ribbon diagrams of a homo-dimer of human (Homo sapiens) caspase-14 (P31944) (https://swissmodel.expasy.org/repository/uniprot/P31944, last accessed on 29 January 2025) and Rhesus macaque (Macaca mulatta) caspase 16 (A0A5F8A9B6) (https://swissmodel.expasy.org/repository/uniprot/A0A5F8A9B6?model=AF-A0A5F8A9B6-F1-model-v4, last accessed on 29 January 2025). Note that an α-helix β-sheet α-helix sandwich fold characteristic for the caspase domain (pfam00656) is predicted for the monomers (turquoise ribbon, monomer A, and gold ribbon, monomer B) of caspase-14 (C) and for both the catalytic domain (upper left) and the prodomain (lower right) of caspase-16 (red ribbon) (D). A subset of residues are labelled. Yellow shading highlights cysteine and histidine residues of the catalytic dyad. The models are reproduced from the SWISS-MODEL repository (Waterhouse et al. 2018) under the CC BY-SA 4.0 Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/) (Color figure online)

Fig. 4

CASP16 has undergone pseudogenization in humans after their evolutionary divergence from other primates. A Schematic depiction of human CASP16P and chimpanzee (chimp) CASP16. Exons are shown as boxes. Protein-coding segments are shaded grey and untranslated regions are white. The position of the frame-shift mutation in human CASP16P and the splicing phases between exons are indicated. B Alignment of nucleotide sequences homologous to the site of the frame-shift mutation in human CASP16P. A phylogenetic tree of the species is shown on the left. GenBank accession numbers: human (Homo sapiens) NC_000016.10, nucleotides 3,144,522–3,144,580; chimpanzee (Pan troglodytes) NC_072416.2, nucleotides 5,813,557–5,813,616; gorilla (Gorilla gorilla gorilla) NC_073242.2, nucleotides 6,791,343–6,791,402; orangutan (Pongo abelii) NC_072003.2, nucleotides 3,319,717–3,319,776